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http://dx.doi.org/10.17576/jsm-2020-4911-12

A Tyrosine-Based Nanosensor for Rapid Sensitive Detection of Copper (II) Ions

(Pengesan Nano Berasaskan Tirosina untuk Pengesanan Sensitif Pantas Ion Tembaga (II)) JIAQI LIAN, PANDENG MIAO, NA LI, ABDUL JAMIL KHAN, XIANG JI* & FENG ZHANG

INTRODUCTION

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ABSTRACT

Most of the chromophores of fluorescent peptides contain aromatic amino acids with conjugated double bonds, among which tyrosine (Y) has become the focus of researches due to its unique physicochemical (optical, redox, and metal chelation) properties. However, there are few studies on the self-assembly and polymerisation of single. This study shows that the phenol group of Y can be oxidized into benzoquinone group in alkaline conditions and then undergoes polymerisation and further self-assembles into nanoparticles (NPs). The product of pYoxNPs have a strong fluorescence emission peak at 463 nm, and Cu2+ can spontaneously bind to it and dramatically quench their fluorescence. Based on these findings, we developed a rapid, sensitive and specific nanosensor for detecting Cu2+. When the concentration of Cu2+ is within the range of 40 μM - 1 mM, we can obtain a good linear correlation between the fluorescence intensity of pYoxNPs and the concentration of copper ions, and the limit of detection (LOD) is determined to 37.26 μM. In comparison to other modern methods for sensing Cu2+, this method has advantages of simplicity of material synthesis, low cost, robust and rapid in sensing reaction, so we envision a good prospect for Cu2+ detection applications in both bulk and harsh environments.

Keywords: Copper ion; fluorescence quenching; oxidation; polymerisation; tyrosine

ABSTRAK

Kebanyakan kromofor peptida pendarfluor mengandungi asid amino aromatik dengan ikatan berganda konjugasi, antaranya tyrosin (Y) telah menjadi tumpuan penyelidikan kerana ciri unik fizikokimianya (optik, redoks dan pengkelatan logam). Walau bagaimanapun, terdapat beberapa kajian mengenai pemasangan diri dan pempolimeran tunggal Y. Dalam kajian ini, kumpulan fenol Y boleh dioksidakan kepada kumpulan benzoquinon dalam keadaan alkali dan kemudian menjalani pempolimeran dan seterusnya menyambung diri ke nanozarah (NPs). pYoxNPs produk mempunyai puncak pelepasan pendarfluor yang kuat pada 463 nm dan Cu2+ secara spontan dapat mengikat mereka dan secara mendadak memadamkan pendarfluor mereka. Berdasarkan penemuan ini, kami membangunkan pengesan nano pesat, sensitif dan khusus untuk mengesan Cu2+. Apabila kepekatan Cu2+ berada dalam julat 40 μM - 1 mM, kita boleh mendapatkan korelasi linear yang baik antara keamatan pendarfluor pYoxNPs dan kepekatan ion tembaga, dan had pengesanan (LOD) ditentukan kepada 37.26 μM. Berbanding dengan kaedah moden yang lain untuk mengesan Cu2+, ini memperlihatkan kelebihan dalam kesederhanaan sintesis bahan, kos rendah dan mudah diperoleh, cepat dan pantas dalam reaksi penderiaan, jadi kami membayangkan prospek yang baik untuk aplikasi pengesanan Cu2+ dalam persekitaran pukal dan mencabar.

Kata kunci: Ion tembaga; pelindapkejutan pendarfluor; pengoksidaan; pempolimeran; tirosina

INTRODUCTION

Copper is one of the most important microelements in human body (Nieder et al. 2018). It mainly participates in the composition of enzymes in the organism and regulates the metabolism of both lipid and sugar (Baker et al. 2017;

Kusunuru et al. 2013). However, excessive copper can cause poisoning (Bulcke et al. 2017).

Moreover, few mental illnesses such as schizophrenia and Alzheimer’s disease (AD) are also related to copper ions (Cu2+) (Brewer 2012; Yu et al. 2018). At present,

the detection technology of Cu2+ mainly includes atomic absorption spectroscopy, colorimetry, fluorescence quenching and electrochemiluminescence analysis (Ghasemian et al. 2012). Among them, fluorescence- based methods attract great interest from researchers due to their outstanding advantages of fast response and high sensitivity. The traditional fluorescence-based methods mainly make use of organic dyes, and metal nanoclusters and inorganic quantum dots as chromophores (Li et al. 2017). Most of these chromophores are

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difficult to synthesize, unstable or toxic, and are not environmentally-friendly (or biocompatible). Contino et al. (2016) used unoxidized L-tyrosine (Y)-coated silver nanoparticles (AgNPs) to detect Cu2+; however, the instability of AgNPs significantly limits its applications due to easy oxidation. Xu et al. (2010) used colorimetric method to detect Cu2+ based on the gold nanoparticle (AuNP)-nucleotide (single strand) complex, in which three designed single-strand DNA can co-assemble to nanostructures, whose melting temperature (Tm) will increase with addition of Cu2+, and this alteration is linear within a certain range of Cu2+ concentrations. However, this approach requires a time-consuming preparation.

White and Holcombe (2007) used the Fmoc-based solid- phase synthesis method to produce peptides modified with a fluorescence chromophore of dansyl chloride, which can detect Cu2+ with their metal ion chelation capacity and then quench the fluorescence upon binding, however, the peptide synthesis is costly. Therefore, it is necessary to develop a new low-cost, simple and environment-friendly method for Cu2+ detection.

Peptide-based nanostructures are sensitive to the environmental conditions such as ionic strength, and temperature. Because of weak-intermolecular interactions such as van der Waals force, hydrophobic interaction, π-π stacking, electrostatic interaction, ionic and hydrogen bonds, their conformational changes will eventually cause the fluctuations of its inherent functionality, which usually is used to design sensors (Parmar et al.

2016). Hence, it would be great to construct fluorescent nanostructures using peptides or amino acids. However, to emit fluorescence in a visible light region is still a big challenge with peptide-based nanostructures. This is because natural amino acids such as phenylalanine (F), Y and tryptophan (W), can only emit fluorescence in the ultraviolet region (Teale & Weber 1957).

Previous studies have found that self-assembly of aromatic amino acids or short peptides can emit high- quantum-yield fluorescence in the visible light region (Pinotsi et al. 2016), in which probabilities for photo- radiation transition of electrons has been enhanced by decreasing the inactivation pathways (like thermal conversions) of activated electrons on the benzene ring, and meanwhile, the self-assembly enlarges the conjugated electron structures (Guo et al. 2019). Among these amino acids, Y has become the focus of researches because of its unique physicochemical properties and its importance in biological metabolism. For example, Y forms catecholamines under the action of enzymes (such as dopamine hydroxylase) in vivo, which has been found closely related to a mental disorder called attention deficit hyperactivity disorder (ADHD) in children (Bergwerff et al. 2016). Y can also form melanin under the action of tyrosinase, and the lack of tyrosinase can cause albinism (Kamaraj & Purohit 2014). At present, there have been many reports on the effects of nitration of Y and the Y residues of proteins, but few studies on the self-assembly and polymerisation of single Y molecules (Bartesaghi &

Radi 2018).

In this study, aiming to build a biocompatible nanosensor for Cu2+ detection, we try to employ both polymerisation and self-assembly to produce fluorescent nanostructures using only Y, and further test it for Cu2+

sensing (Lin et al. 2011). In practice, we found that Y can be oxidized to its oxidized state Yox in alkaline solutions and further form polymer nanoparticles (pYoxNPs) by both polymerisations of Yox and self-assembly, and the pYoxNPs can emit stable and strong fluorescence, which can apply to sense Cu2+ due to their efficient quenching action (Figure 1).

FIGURE 1. A schematic illustration of pYoxNP construction and Cu2+ sensing. pYoxNPs form by a combination of oxidation, polymerisation, and self-assembly in alkaline

conditions. The purple arrows represent ultraviolet light, and the green ‘thunder’

represents fluorescence as excited by ultraviolet light. The blue fluorescence of pYoxNPs can apply to Cu2+ sensing based-on an efficient quenching mechanism

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MATERIALS AND METHODS MATERIALS AND APPARATUSES

L-Tyrosine and all other reagents such as CuCl2, CaCl2, CdCl2, MgCl2, LiCl, NiCl2, PbCl2, ZnCl2, NaCl, and AgNO3 metal ions were supplied by Aladdin Reagent Co. Ltd. (Shanghai, China); NaOH,HCl was supplied by Beijing Chemical Reagent Co. Ltd. (Beijing, China). All the metal ion compounds are analytical reagent grade without further purification. Deionized water (18.2 MΩ∙cm) used for all experiments was made from a Milli-Q system (Millipore, Bedford, USA). All solvents were deionized water except for special descriptions. UV- Vis spectrometer (U-2900, Hitachi, Japan), Fluorescence spectrometer (Fluorolog®-MAX 4, Horiba, Japan), dynamic light scattering (DLS) equipment (Malvern Zetasizer Nano-ZS90, Malvern Instruments Ltd., UK).

PREPARATION OF pYoxNPs

Dissolved 72 mg of tyrosine in 20 mL of sodium hydroxide (0.1 M) and 20 mL of hydrochloric acid (0.1 M), respectively. Weighed 7.2 mg of tyrosine and dissolved it in 20 mL of water, heated it at 80 °C for condensation and refluxed for 4 h, then stopped heating and continue stirring overnight. After filtration with 0.22 μM membrane filters, we got the reserve solution of tyrosine dissolved in sodium hydroxide solution, i.e. synthetic polymerised oxidized Y NPs (pYoxNPs), tyrosine hydrochloric acid solution reserve solution (Y-HCl), and tyrosine aqueous solution reserve solution (Y-H2O), which was stored at room temperature. In addition, the particle size was measured by the DLS equipment (DLS), and the absorbance of the solution was measured with the ultraviolet visible spectrometer, the fluorescence spectra was recorded with the fluorescence spectrometer, and the solution fluorescence was observed under the UV lamp.

ASSAYS FOR SENSING Cu2+

Weighed 85.2 mg of cupric chloride dihydrate dissolved in 5 mL water to prepare a Cu2+ stock solution (100 mM), then gradually dilute it to obtain a concentration gradient as 10000, 7500, 5000, 2500, 1000, 750, 500, 250, 100, 75, 50, 25 μM. Took 100 μL of each gradient solution into the centrifuge tube and added 900 μL of oxidized tyrosine (Yox) stock solution, respectively, mixed and standed for 2 h. To measure fluorescence, the samples were excited at 365 nm, and the standard curve was made by plotting the fluorescence intensity at 463 nm against Cu2+ concentration. In order to determine the specificity of the method, we designed the interference experiment of other metal ions, and prepared metal ions solutions, such as: Zn2+, Pb2+, Ni+, Na+, Mg2+, Li+, Cd2+, Ca2+, Ag+ (100 μm). By treating these ions with the same method

for Cu2+, we can analyze the ‘interference’ of these ions on fluorescence. The limit of detection (LOD) was calculated according to the 3σ method.

RESULTS AND DISCUSSION CHARACTERIZATION OF pYoxNPs

To study the physicochemical properties, we obtained three Y-containing stock solutions, termed as pYoxNPs, Y-HCl, and Y-H2O. The stock solution of pYoxNPs exhibits yellow colour, while stock solutions of Y-H2O and Y-HCl are colourless. It is worth noting that the concentration of Y in water cannot be comparable to those in HCl and NaOH solutions, so to prepare Y-H2O solution must be under the conditions of both heating and stirring overnight, the low water-solubility of Y is due to its natural chemical property (Li et al. 2019). In contrast, the preparation of Y-NaOH solution requires neither heating nor stirring overnight. For the other two solutions Y-H2O and Y-HCl, a single peak at 274 nm shows in their absorption spectra (Figure 2(A)), indicating that Y is not oxidized. However, in the alkaline solution, the single absorption peak shifts to 293 nm and became bigger (Figure 2(A)), indicating the chemical changes of Y molecules. In terms of solution colour, the unheated Y-HCl solution is colourless and transparent, however, pYoxNPs solution is yellow though still transparent, which could be due to the oxidation of hydroxyl groups on benzene ring under heating conditions, resulting in the change of liquid colour (Figure S1). In terms of molecular structure, deprotonation occurs to Y in alkaline solutions, producing groups like -COO- and -NH- (Figure S2), both of which enhance the solubility of Y. The blue fluorescence of pYox was observed using an excitation at 365 nm. The maximum emission peak was found at 463 nm in alkaline solutions (pYox), but not in the control solutions like Y-H2O, Y-HCL and Y-NaOH (Figure 2B). We speculate that a new additional ring (indoline structure) formed on the benzene ring of Y (Figure S2), creating a novel chromophore which makes the emission wavelength red-shifted (Lee et al. 2016).

According to the principle of fluorescence (Verlag 2006), some ground-state electrons outside the nucleus can be excited by the high energy of light, and then electron transition occurs. When this excited electron returns to the ground state, the energy will be released in the form of radiation to produce fluorescence. But not all energy can be released in the form of radiation, usually a small fragment of energy is released in the form of non-radiation like heat. When a single Y molecule absorbs UV light and its electrons firstly excited to high-energic orbitals and then they return to the ground state, some energy of the excited electrons will inevitably lose in a non- radiative transition way due to the rotational vibration of

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the benzene ring, which will hardly produce fluorescence.

Similar to GFP (Tsien 1998), the chromophore of GFP needs a β-barrel cage to lock its chromophore’s rotation to improve its fluorescence quantum yield and meanwhile prevent water from quenching its fluorescence utilizing spatial isolation. After the cyclization and oxidation of Y residues, new chromophores were formed (Figure S2), which can be verified by the optical changes (Figure 2).

After the oxidation and cross-linking of benzene rings (Figure S2), the chromophores were further locked to prevent rotating. All excited electrons should release energy via increased radiation-based transition, thus reduction radiation-based transition, eventually save energy for producing fluorescence (Figure 2) (Ren et al.

2019).

In order to proves that Y occurred crosslinking and polymerisation, DLS measurement was carried out.

We observed that the hydrodynamic size of pYoxNPs was about 5 nm, which is with following the normal distribution (Figure 3). If we increased the reflux time, we found that the hydrodynamic size pYox gradually increased (data not shown), indicating the extent of polymerisation/crosslinking. However, no matter if the reflux time is long or short, the DLS cannot obtain a good result for Y dissolved in HCl (Y-HCl) or water (Y-H2O).

However, because these solutions (Y-HCl and Y-H2O) are colourless and transparent (Figure S1), indicating that Y is still dispersed in the solvent as a single molecule.

Thus, the molecule is too small to be measured by the current DLS instrument, which further proves that Y does not occur oxidation and the successive cross-linking/

polymerisation in acidic and neutral solutions.

FIGURE 2. Optical characterization of Y under different conditions. Absorption A) and photoluminescence (PL).

B) spectra of as-prepared Y samples in different conditions. pYox, the polymer of oxidized Y; Y-NaOH, the control solution of Y dissolved in diluted NaOH (without heating and reflux); pYox+ Cu2+, polymer of oxidized Y mixing with Cu2+; Y-H2O, Y just dissolved in water; Y-HCl, Y dissolved in dilute HCl solution; the detailed preparation can refer

to the experimental section

FIGURE 3. DLS analysis of the hydrodynamic size distribution of pYox before and after mixing with copper ions. The blue and green histograms represent pYox and pYox + Cu2+ samples, respectively. The red lines are the single peak fitting

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ASSAYS FOR SENSING Cu2+

pYoxNPs emit blue fluorescence, which can be efficiently quenched upon binding with Cu2+ (pYox+ Cu2+, Figure 2(B)). The size distribution of pYoxNPs+ Cu2+ is measured to about 30 nm, which is significantly larger than that of pYoxNPs themselves (Figure 3). With a new absorption peak appearing at 329 nm, we deduce that the cation ions can strongly coordinate with pYoxNPs (Figure 2(A)). When the Cu2+ concentration is within the range of 25 μM - 10 mM, the fluorescence of pYoxNPs at 463 nm decreases along with the increasing of Cu2+ concentration (Figure 4(A)). The plot of fluorescence intensity against the concentration of Cu2+ shows a shape of decay curve, so it was well fitted to an exponential decay function (Figure 4(B)) with the formula shown in (1) and (2).

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(2) When Cu2+ concentration ranges from 40 μM to 1 mM, the fluorescence intensity of pYoxNPs changes linearly with Cu2+ concentration (Figure 5(A), S3(A)), with a linear equation, as shown in (3).

(3) The limit of detection (LOD) calculated by 3σ rule is 37.26 mΜ, which represents a relatively high sensitivity to Cu2+. In addition, the Cu2+ quenching of the fluorescence of pYoxNPs is also consistent with the Stern-Volmer equation (Figure S2), indicating a static quenching mechanism further proves the robust coordination between pYoxNPs and Cu2+.

𝑦𝑦 = 2.5 × 105− 3.4 × 104× 𝑒𝑒1.3×102𝑡𝑡 + 2.5 × 105× 𝑒𝑒1.0×103𝑡𝑡 + 3.4 × 105× 𝑒𝑒4.9×103𝑡𝑡 (1)

𝑡𝑡 = 0.6 − 𝑥𝑥 (2)

𝑦𝑦 = 2.5 × 105− 3.4 × 104× 𝑒𝑒1.3×102𝑡𝑡 + 2.5 × 105× 𝑒𝑒1.0×103𝑡𝑡 + 3.4 × 105× 𝑒𝑒4.9×103𝑡𝑡 (1)

𝑡𝑡 = 0.6 − 𝑥𝑥 (2)

𝑦𝑦 = 2.5 × 105− 3.4 × 104× 𝑒𝑒1.3×102𝑡𝑡 + 2.5 × 105× 𝑒𝑒1.0×103𝑡𝑡 + 3.4 × 105× 𝑒𝑒4.9×103𝑡𝑡 (1)

𝑡𝑡 = 0.6 − 𝑥𝑥 (2)

y = −209.44𝑥𝑥 + 8.6 × 105 (R2= 0.9916)

FIGURE 4. Sensing assays A) Fluorescence spectra of pYoxNPs solutions mixed with gradient concentrations of Cu2+ as indicated in the figure (from upper to bottom, the concentration is increased), B) A plot of pYoxNPs fluorescence intensity at 463 nm against Cu2+ concentration (black dots), and the corresponding fitting with a correlation coefficient (R2) of 0.9989

Taking other metal ions and Cu2+ of the same concentration for a comparison measurement, we analysed the interference of other metal ions on this method. The interference experiments of various common metal ions (100 μM) on the detection system showed that only Cu2+

had the best fluorescence quenching effect on pYoxNP solution, while the other cations showed little effects on pYoxNPs solution (Figure 5(B)), indicating a relatively high specificity for binding with Cu2+.

Compared with other aromatic amino acids as fluorescent chromophores, the dopa quinone formed by oxidation of Y residues has redundant lone-pair electrons and is more likely to chelate metal ions (Lee & Lee 2015). Moreover, after crosslinking/polymerisation, the fluorescence of pYox solution exhibited a redshift, which could be due to the interactions of both hydrophobicity and π-π stacking. It is found that Cu2+ cannot make the pYoxNPs aggregate and thus increase their hydrodynamic

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sizes, but also strongly quench their fluorescence, which could be explained by the negative value of the Gibbs free energy ΔG (-16.27kJ/mol, see SI), indicating a

At last, we also assessed the reliability of the current developed approach, and the results are displayed in Table 1. By analysing the recoveries of Cu2+, which varied from 104% to 112% with a relative standard deviation (RSD) ranging from 0.36 to 0.90, we think the results achieved

spontaneous binding (Liu et al. 2015; Yuan et al. 2016;

Zhong et al. 2014) between Cu2+ and pYoxNPs.

FIGURE 5. The optimized concentration ranges for sensing Cu2+ and the selectivity comparison of cations A) The plot of pYoxNP fluorescence intensity at 463 nm against the Cu2+ concentration (black dots), and the corresponding linear fitting (red line) with a function and the correlation coefficient (R2), B) The selectivity or interference test of cations for pYoxNPs-based sensor. I0 and I represent the 463 nm fluorescence intensity of pYoxNPs in the absence and

presence of cations

by our method are in good agreement with the real values, indicating that the current sensing technology reported in this article are robust and reliable for sensing Cu2+ in aqueous solutions.

TABLE 1. Reliability evaluation of the Cu2+ sensing

Cu2+ samples Added Cu2+ (µM) Detected Cu2+ (µM) RSD (n=5, %) Recovery (%)

1 250 272.06 0.90 108.82

2 500 559.53 0.36 111.91

3 750 780.44 0.39 104.06

CONCLUSION

New technologies can inevitably innovate the living ways such as the clinical diagnosis in the contemporary era. The current fluorescent copper ion nanosensor was constructed via the combination of oxidation,

polymerisation and self-assembly of single Y residues in basic conditions. We envision that co-assembling of more amino acids might create more versatile functional nanostructures, which hold great potential in bio-labeling/

imaging/sensing, and even therapy for diseases. We also

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Proteomic alterations of brain subcellular organelles caused believe this study will inspire scientists to open a new

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ACKNOWLEDGEMENTS

The following programs and foundations supported this work: the University Scientific Research Project of Inner Mongolia Autonomous Region (NJZC17267), the Program Funded by University for Fostering Distinguished Young Scholars, the National Natural Science Foundation of China (No.51763019, U1832125), the Grassland Talents Program of Inner Mongolia Autonomous Region, the Distinguished Young Scholars Foundation of Inner Mongolia Autonomous Region, and the Young Leading Talents of Science and Technology Program of Inner Mongolia Autonomous Region.

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Jiaqi Lian, Xiang Ji1* & Feng Zhang*

School of Life Science and Technology

Inner Mongolia University of Science and Technology Baotou 014010

P. R. China

Jiaqi Lian, Pandeng Miao, Na Li, Abdul Jamil Khan & Feng Zhang*

Biomedical Nanocenter, School of Life Science Inner Mongolia Agricultural University Hohhot 010018

P. R. China

Pandeng Miao & Na Li

Terahertz Technology Innovation Research Institute Shanghai Key Laboratory of Modern Optical System Terahertz Science Cooperative Innovation Center University of Shanghai for Science and Technology 516 Jungong Road, Shanghai 200093

P. R. China Feng Zhang*

Key Laboratory of Oral Medicine Guangzhou Institute of Oral Disease

Stomatology Hospital, Department of Biomedical Engineering School of Basic Medical Sciences

Guangzhou Medical University Guangzhou 511436

P. R. China

*Corresponding author; email: jixiang@imust.cn Received: 17 January 2020

Accepted: 8 June 2020

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FIGURE S1. The color comparison of Y-HCl, Y-H2O and pYox solutions (from left to right) under daylight

FIGURE S2. A chemical evolution procedure of Y in alkaline solutions. In sodium hydroxide solution, Y can be firstly deprotonated to produce imine group (-NH-), deprotonated hydroxyl

group (-O-) and carboxylic group (-COO-), and then the imine group and benzene ring can cyclize to form a two-ring structure (indoline group, whose absorption peak shifts to red region), then hydroxyl groups are further oxidized to form O-benzoquinone (Yox), which eventually

polymerize to pYox by cross-linking reaction (Ding et al. 2015)

FIGURE S3. The assays of pYoxNPs, fluorescence quenched by Cu2+ A). The steady-state fluorescence spectra of pYoxNPs mixed with various concentrations of Cu2+ ranging from 0 to 1000 µM as indicated by the arrow in the figure. The spectra were recorded at 298 K with an excitation of 365 nm, B) The

Stern-Volmer plot (black dots) for the fluorescence quenching ratio of pYox against Cu2+ ion (black dots) and the corresponding linear fitting (red line). I0 and I represent the fluorescence intensity of the

pYoxNP solution before and after mixing with different concentrated Cu2+

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FLUORESCENCE QUENCHING AND THERMODYNAMICS

In order to figure out the mechanism how Cu2+ can quench pYoxNPs, we studied the thermodynamics of this binding system, in which the quencher is Cu2+. According to the Stern-Volmer equation (Ghosh & Chattopadhyay 2015) (1), in which I0 and I are the fluorescence intensities in presence and absence of quenchers, respectively.

[Q], KSV and τ0 are the concentration of quencher, the quenching constant of Stern-Volmer and the life time of fluorescence, respectively.

(S1)

(S2) Generally, the quenching constant Kq is used to better describe this process. And all the τ0 of biomolecules can be considered as 10-8 s, therefore, if using the quenching constant (Kq, (2)) to replace KSV in (1), we can obtain KSV

= 3.1 × 102 M−1 and Kq = 3.1 × 1010 M−1. S−1. Because the 𝐼𝐼𝐼𝐼 0 = 𝐾𝐾𝑆𝑆𝑉𝑉[𝑄𝑄] + 1 (S1)

𝐾𝐾𝑆𝑆𝑉𝑉 = 𝐾𝐾𝑞𝑞 × 𝜏𝜏0 (S2) 𝐼𝐼𝐼𝐼 0 = 𝐾𝐾𝑆𝑆𝑉𝑉[𝑄𝑄] + 1 (S1)

𝐾𝐾𝑆𝑆𝑉𝑉 = 𝐾𝐾𝑞𝑞 × 𝜏𝜏0 (S2)

log[𝐼𝐼0− 𝐼𝐼 𝐼𝐼 − 𝐼𝐼𝑠𝑠𝑠𝑠𝑠𝑠]=log 𝐾𝐾𝑠𝑠+ 𝑛𝑛log[𝑄𝑄]

Kq is bigger than the diffusion-controlled limit (normally near 1 × 1010 M−1. S−1), therefore, we think pYoxNPs and Cu2+ involve static binding.

In order to verify this reaction can occur spontaneously, we also calculate the Gibbs free energy ΔG (Hemmateenejad & Yousefinejad 2013). By using the Hill equation (S3) in which Isat, η and Kα refer to the fluorescence intensity of pYoxNPs mixed with a saturated amount of Cu2+, Hill coefficient and the binding constant, respectively.

(S3)

The Kɑ was calculated to 7.1 × 102 (M-1), with which the

∆G can be calculated to −1.6 × 101kJ∙mol-1 (S4).

∆G = −RT ln Kɑ (S4) Because the ∆G < 0, therefore, the binding between Cu2+

and pYoxNPs should be spontaneous.

Rujukan

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